In order to identify genes that normally respond to insulin in vivo and specifically, to explore the pathway of insulin regulated gene expression in human skeletal muscle in individuals with different insulin sensitivities, we have adapted the S1 nuclease protection assay for use with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from multiple genes. In conjunction with hyperinsulinemic, euglycemic clamp technique, we have evaluated the ability of insulin to alter the RNA abundances of several proto-oncogenes and on other potentially insulin responsive genes in human skeletal muscle from individuals with differing insulin sensitivities over a two hour time period. Of the 9 genes examined with this technique, 4 proved to be insulin responsive in vivo in insulin sensitive individuals. The proto-oncogenes c-Ha-ras, c-myc, and c-src all displayed a 2-4 fold transient increases in their respective RNA levels within 30 minutes of insulin infusion. In addition, myf-5, a muscle specific differentiation factor also proved to be insulin sensitive. The abundance of this RNA also increased 3-fold with a time course similar to those displayed by c- Ha-ras and c-src. The responses of c-Ha-ras and myf5 were diminished in insulin resistant individuals. C-myc, while responding to insulin stimulation in both groups showed overall lower levels in individuals with decreased insulin sensitivity. In contrast, c-src RNA levels increased in response to insulin in both groups. While RNA abundance of c-jun and insulin receptor were not altered over the two hour time course of insulin administration, the basal RNA levels were lower in individuals that are insulin resistant. This suggests that there are multiple insulin signal pathways that result in modifications in gene expression. Glut-3, Glut-4 and c-fos showed neither statistically significant increases in RNA levels nor were basal RNA levels altered by decreased insulin sensitivity.